In animals the timekeeping necessary for sleep/activity rhythms takes place within neurons located in discrete regions of the central nervous system (CNS). Remarkably, individual neuronal circadian clocks can maintain molecular and physiological rhythms in the absence of time-cues from the environment or other cells. Despite such cell-autonomous timekeeping, the orchestration of daily behavioral rhytiims depends on networks of clock neurons. I seek to understand the circuit properties of these networks and to examine the roles that identified neurons play in the control of circadian rhythms. During the ROO phase of my research! will use the anatomical, genetic, and live-imaging techniques 1 developed during the K99 phase of my grant to discover how time is kept within the brain and how it is used to orchestrate daily and seasonal changes in behavior in the fly Drosophila melanogaster. Furthermore, newly developed technologies in live imaging, fly genetics, and light-control of neuronal signaling will be developed to investigate the circuit properties of the circadian clock network in the fly brain. To understand the organization of the neuronal clock network, and fundamental questions of neuromodulatory signaling in the nervous system, I propose the following speciflc aims: 1) The development of live Imaging methods for the simultaneous manipulation and observation of neuronal signaling using optogenetic control of cell excitability in conjunction with genetically encoded sensors for cAMP and Ca2-f, H) The identiflcation of peptide/amine modulators of the neuronal clock network. Ill) The identiflcation and characterization of non-clock targets of clock neuron output using live imaging and targeted genetic manipulation approaches, IV) The genetic dissection of GPCR/cAMP/Ca2-t- signaling within these neurons. The work supported by my K99 grant supports the feasibility of all aspects of the proposed research and the resources made possible by the ROO will allow me to employ advanced imaging technologies in my study of the neuronal clock network. Furthermore, the work proposed here will address fundamental aspects of neurobiology and create new methodologies for the investigation of neuronal circuitry and the control of animal behavior.